Note: Descriptions are shown in the official language in which they were submitted.
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USE OF ADJUVANTS FOR THE PREVENTION AND/OR TREATMENT OF AUTOIMMUNE
DISEASES
Technical field
The present invention relates to adjuvants, in particular to the use of
adjuvants in the prevention and/or
treatment of autoimmune diseases.
Background of the invention
In many vaccines, antigens are in the form of purified or subunit proteins to
improve their safety,
but this comes at the expense of lower immunogenicity. Therefore, effective
adaptive immune responses
require the addition of adjuvants to trigger early inflammation and activate
antigen presenting cells.
Aluminium adjuvant (alum) is widely used to promote antibody responses by
inducing a Th2
response. Squalene-based adjuvants MF59 and adjuvant system (AS) 03 promote
both Th1 and Th2
responses through their capacity to induce chemokine and cytokine release,
leading to massive recruitment
and activation of immune cells. As for the Toll-like-receptor ligand-based
adjuvants A504 and A501, they
induce Th1 and Th2 responses by directly activating innate immune cells.
Beyond the effects on effector responses, some studies revealed intriguing and
paradoxical
observations of the immunomodulatory impact of certain adjuvants in autoimmune
diseases. Administration
of complete Freund's adjuvant (CFA), used only in veterinary vaccines, induced
protection and remission
of type 1 diabetes in non-obese diabetic mice (Sadelain etal. (1990) Diabetes
39: 583). Also, administration
of lipopolysaccharides (LPS), a natural TLR4 ligand used in research trials
for veterinary vaccines, induced
protection from experimental autoimmune encephalomyelitis (EAE), a mouse model
of multiple sclerosis
(Buenafe etal. (2007) J. Neuroimmunol. 182: 32). While these are interesting
observations, both CFA and
LPS are not suitable for human use because of toxicity.
Autoimmune diseases are caused by an immune response against constituents of
the body's own
tissues. More than 80 autoimmune diseases are known. Examples of autoimmune
diseases include e.g.
rheumatoid arthritis (RA), systemic lupus erythematosus (lupus), inflammatory
bowel disease (IBD),
multiple sclerosis (MS), type 1 diabetes mellitus, Guillain-Barre syndrome,
Crohn's disease and psoriasis.
Many of these diseases are chronic and can cause significant morbidity and
disability. Treatment of
autoimmune diseases is generally based on immunosuppression. While significant
progress has been made
in the treatment of autoimmune diseases, there is still a need for improved
products and methods, which
increase efficacy, reduce side-effects, are easy to administer, safe and can
be used in long-term or even
chronic treatment of these diseases.
Summary of the invention
It has now surprisingly been found that AS01 and A503, two non-alum containing
adjuvants suitable for,
and approved for, human use, can induce almost complete prevention of
autoimmune disease in an animal
model, supporting their use in the prevention and/or treatment of autoimmune
diseases in humans.
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Accordingly, in a first aspect, the invention relates to an adjuvant for use
in the prevention and/or
treatment of an autoimmune disease, wherein the adjuvant does not comprise
aluminium and is suitable
for use in a human subject.
In a further aspect, there is provided a method for preventing and/or treating
an autoimmune
disease comprising administration of an adjuvant to a subject, wherein the
adjuvant does not comprise
aluminium and is suitable for use in a human subject.
In an even further aspect, the invention relates to the use of an adjuvant in
the manufacture of a
medicament for the prevention and/or treatment of an autoimmune disease,
wherein the adjuvant does
not comprise aluminium and is suitable for use in a human subject.
Brief description of the figures
Figure 1: a, Representative picture of popliteal draining lymph nodes (dLN) 4
days after subcutaneous
injection (footpads) of vaccine adjuvants. b, Typical flow cytometry analysis
of Treg cells. c, Fold-increase
of the percentage of Foxp3+ cells among CD4+ cells, compared to mice injected
with PBS, in pLN 4 days
and 7 days after subcutaneous injection of vaccine adjuvants. Cumulative data
from 3 independent
experiments. d, CD4+Foxp3-CD90.1+ cells were adoptively transferred prior to
adjuvant injection and
induction of Foxp3 on donor cells was evaluated at day 4 as a marker of pTreg
cells. Represented is an
example of analysis of Treg induction following AS01 treatment. e,f, Ex vivo
suppressive activity of Treg
cells purified from dLN of mice treated with AS01 4 days earlier.
Representative data at a 1 to 2 Treg:Tconv
cell ratio (e) and mean SEM at different Treg:Tconv cell ratio from 4
independent experiments M. "Treg"
cells is for regulatory T cells and "Tconv" cells is for conventional T cells.
Figure 2: a, Absolute number of CD45+ cells in dLN 4 days after subcutaneous
injection of vaccine
adjuvants. Cumulative data from 3 independent experiments. b, Representative
expression of CD44, ICOS
and KI-67 in Treg cells from dLN 4 days after subcutaneous injection of AS01.
c, In vitro suppressive
activity of Treg cells purified from dLN 3 days after subcutaneous injection
of vaccine adjuvants. d,
Representative Foxp3 expression in Treg cells from dLN 3 days after
subcutaneous injection of AS01. e, In
vitro suppressive activity of Treg cells purified from dLN 7 days after
subcutaneous injection of AS01. "Treg"
cells is for regulatory T cells.
Figure 3: a, Clinical score of EAE (experimental autoimmune encephalomyelitis)
in mice immunized to
induce EAE at day 0 and treated with adjuvants at day -3 and 0. Cumulative
data from 3 independent
experiments. b, Representative proliferation (from 2 independent experiments)
of 2D2 M0G35-55-specific T
cell 3 days after transfer in mice immunized to induce EAE (day 0) and treated
with adjuvants (day -3). C,
INF¨y and IL-17 production by MOG-reactive T cells from dLN of mice immunized
10 days earlier to induce
EAE (day 0) and treated with A501 or A503 (day -3). d, Clinical score of EAE
in mice immunized to induce
EAE at day 0 and transferred the day before with Treg cells purified from PBS,
A501 or A503 treated mice.
e,f Percentage of Treg cells (e) and of integrin a L+ and a M+ among Treg and
Tconv cells (f) in dLN at
day 10 in mice immunized to induce EAE at day 0 and treated with A503 at day -
3. "Treg" cells is for
regulatory T cells and "Tconv" cells is for conventional T cells.
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Figure 4: a, b Clinical score of EAE (experimental autoimmune
encephalomyelitis) in mice immunized to
induce EAE at day 0 and treated with A501 or AS03 at day -3 (a) or day 0 (b).
Cumulative data from 3
independent experiments. c,d Proportions of CCR6+ cells and CXCR3+ cells (c)
and of integrin a 4+ cells
and integrin a L+ cells (d) among Treg cells and Tconv cells in dLN at day 10
in mice immunized to induce
EAE at day 0 and treated with AS03 at day 0 and -3. "Treg" cells is for
regulatory T cells and "Tconv" cells
is for conventional T cells.
Detailed description
As described above, in a first aspect, the invention relates to an adjuvant
for use in the prevention
and/or treatment of an autoimmune disease, wherein the adjuvant does not
comprise aluminium and is
suitable for use in a human subject.
Typically, the aim of the use of the invention, or of the method of the
invention, is to prevent an
autoimmune disease, including delaying onset of the disease, and/or treat such
a disease, i.e. reduce the
severity of such a disease, e.g. by reducing the cause of the autoimmune
disease and/or reducing its
symptoms. In one embodiment, a reduction of symptoms of more than 50%, such
more than 75%, as
determined according to the Examples herein, is achieved.
Adjuvants for use in the invention
In some embodiments, the adjuvant comprises an immunologically active saponin,
e.g. Q521.
Adjuvants comprising saponins have been described in the art. Saponins are
e.g. described in: Lacaille-
Dubois and Wagner (1996) (A review of the biological and pharmacological
activities of saponins.
Phytomedicine Vol. 2: 363). Saponins are known as adjuvants in vaccines. For
example, Quil A (derived
from the bark of the South American tree Quillaja Saponaria Molina), was
described by Dalsgaard et al.
(1974) in "Saponin adjuvants" (Archly. fur die gesamte Virusforschung, Vol.
44, Springer Verlag, Berlin,
243) to have adjuvant activity. Purified fractions of Quil A have been
isolated by HPLC which retain adjuvant
activity without the toxicity associated with Quil A (Kensil et al. (1991) J.
Immunol. 146: 431). Quil A
fractions are also described in US 5,057,540 and "Saponins as vaccine
adjuvants" (Kensil, C. R., Crit. Rev.
Ther. Drug Carrier Syst., 1996, 12 (1-2): 1-55).
Two such fractions, suitable for use in the present invention, are Q57 and
QS21 (also known as
QA-7 and QA-21). QS21 is a preferred immunologically active saponin fraction
for use in the present
invention. Q521 has been reviewed in "Vaccine Adjuvants: preparation methods
and research protocols"
(Humana Press, Totowa, New Jersey, Edited by Derek T. O'Hagan, Chapter 15:
"QS21 Adjuvant").
Particulate adjuvant systems comprising fractions of Quil A, such as Q521 and
Q57, are e.g. described in
WO 96/33739, WO 96/11711 and WO 07/068907.
In addition to the saponin component, the adjuvant preferably comprises a
sterol. The presence
of a sterol may further reduce reactogenicity of compositions comprising
saponins, see e.g. EP0822831.
Suitable sterols include beta-sitosterol, stigmasterol, ergosterol,
ergocalciferol and cholesterol. Cholesterol
is particularly suitable. Suitably, the immunologically active saponin
fraction is Q521 and the ratio of
Q521:sterol is from 1:100 to 1:1 w/w, such as from 1:10 to 1:1 w/w, e.g. from
1:5 to 1:1 w/w.
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In some embodiments, the adjuvant used in the invention comprises a TLR (Toll-
like receptor)
agonist, such as a TLR4 agonist, e.g. 3D-MPL. The use of TLR agonists in
adjuvants is well-known in art
and has been reviewed e.g. by Lahiri et al. (2008, Vaccine 26: 6777). TLRs
that can be stimulated to
achieve an adjuvant effect include TLR2, TLR4, TLR5, TLR7, TLR8 and TLR9.
TLR2, TLR4, TLR7 and TLR8
agonists, particularly TLR4 agonists, are preferred.
Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl
lipid A (MPL) and 3-
0-deacylated monophosphoryl lipid A (3D-MPL). US 4,436,727 discloses MPL and
its manufacture. US
4,912,094 and reexamination certificate B1 4,912,094 discloses 3D-MPL and a
method for its manufacture.
Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a synthetic lipid
A-like molecule (see e.g. Fox
et al. (2012) Clin. Vaccine Immunol. 19: 1633). In a further embodiment, the
TLR4 agonist may be a
synthetic TLR4 agonist, such as a synthetic disaccharide molecule, similar in
structure to MPL and 3D-MPL,
or may be synthetic monosaccharide molecules, such as the aminoalkyl
glucosaminide phosphate (AGP)
compounds disclosed in, for example, WO 98/50399, WO 01/34617, WO 02/12258, WO
03/065806, WO
04/062599, WO 06/016997, WO 06/12425, WO 03/066065, and W001/90129. Such
molecules have also
been described in the scientific and patent literature as lipid A mimetics.
Lipid A mimetics suitably share
some functional and/or structural activity with lipid A, and in one aspect are
recognised by TLR4 receptors.
AGPs as described herein are sometimes referred to as lipid A mimetics in the
art. In a preferred
embodiment, the TLR4 agonist is 3D-MPL. TLR4 agonists, such as 3-0-deacylated
monophosphoryl lipid A
(3D-MPL), and their use as adjuvants in vaccines has e.g. been described in WO
96/33739 and WO
07/068907 and reviewed in Alving etal. (2012, Curr. Opin. in Immunol. 24:
310).
In a preferred embodiment of the invention, the adjuvant comprises both an
immunologically active
saponin and a TLR4 agonist, e.g. Q521 and 3D-MPL.
In a further preferred embodiment, the adjuvant comprises an immunologically
active saponin and
a TLR4 agonist, e.g. Q521 and 3D-MPL, in a liposomal formulation.
The term "liposome" when used herein refers to uni- or multilamellar lipid
structures enclosing an
aqueous interior. Liposomes and liposome formulations are well known in the
art. Liposomal presentations
are e.g. described in WO 96/33739 and WO 07/068907. Lipids which are capable
of forming liposomes
include all substances having fatty or fat-like properties.
Liposome size may vary from 30 nm to several pm depending on the phospholipid
composition and
the method used for their preparation. In particular embodiments of the
invention, the liposome size will
be in the range of 50 nm to 500 nm, and in further embodiments, in the range
of 50 nm to 200 nm.
Dynamic laser light scattering is a method used to measure the size of
liposomes well known to those
skilled in the art.
In a particularly suitable embodiment, liposomes used in the invention
comprise DOPC and a sterol,
in particular cholesterol. Thus, in a particular embodiment, adjuvants of the
invention comprise Q521 in
any amount described herein in the form of a liposome, wherein said liposome
comprises DOPC and a
sterol, in particular cholesterol.
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In one embodiment, the adjuvant comprises between 5 and 100, such as between
10 and 75, e.g.
25 or 50 pg, of Q521 per dose and between 5 and 100, such as between 10 and
75, e.g. 25 or 50 pg of
3D-MPL per dose.
It is well known that for parenteral administration, solutions should be
physiologically isotonic (i.e.
have a pharmaceutically acceptable osmolality) to avoid cell distortion or
lysis. An "isotonicity agent" is a
compound that is physiologically tolerated and imparts a suitable tonicity to
a formulation (e.g.
immunogenic compositions of the invention) to prevent the net flow of water
across cell membranes that
are in contact with the formulation. Aqueous adjuvant compositions are known
which contain 100 mM
sodium chloride or more, for example adjuvant system A (AS) in WO 05/112991
and WO 08/142133 or the
liposomal adjuvants disclosed in WO 07/068907.
In some embodiments, the isotonicity agent used for the composition is a salt.
In other
embodiments, however, the composition comprises a non-ionic isotonicity agent
and the concentration of
sodium chloride or the ionic strength in the composition is less than 100 mM,
such as less than 80 mM,
e.g. less than 30 mM, such as less 10 mM or less than 5 mM. In a preferred
embodiment, the non-ionic
isotonicity agent is a polyol, such as sorbitol. The concentration of sorbitol
may be e.g. between about 3%
and about 15% (w/v), such as between about 4% and about 10% (w/v). Adjuvants
comprising an
immunologically active saponin fraction and a TLR4 agonist wherein the
isotonicity agent is salt or a polyol
have been described in WO 10/142685, see e.g. Examples 1 and 2 in WO
10/142685.
In one embodiment, the adjuvant used in the invention comprises an oil-in-
water emulsion.
Suitably, said emulsion comprises a metabolisable oil in an amount of 0.5% to
20% of the total volume,
The meaning of the term metabolisable oil is well known in the art.
Metabolisable can be defined as 'being
capable of being transformed by metabolism' (Dorland's Illustrated Medical
Dictionary, W.B. Sanders
Company, 25th edition (1974)). The oil may be any vegetable oil, fish oil,
animal oil or synthetic oil, which
is not toxic to the recipient and is capable of being transformed by
metabolism. Nuts, seeds, and grains
are common sources of vegetable oils. Synthetic oils are also part of this
invention and can include
commercially available oils such as NEOBEEC) and others. A particularly
suitable metabolisable oil is
squalene. Squalene (2,6,10,15,19,23-Hexamethy1-2,6,10,14,18,22-
tetracosahexaene) is an unsaturated oil
which is found in large quantities in shark- liver oil, and in lower
quantities in olive oil, wheat germ oil, rice
bran oil, and yeast, and is a particularly suitable oil for use in this
invention. Squalene is a metabolisable
oil by virtue of the fact that it is an intermediate in the biosynthesis of
cholesterol (Merck index, 10th
Edition, entry No. 8619).
Oil-in-water emulsions per se are well known in the art, and have been
suggested to be useful as
adjuvant compositions (EP 399843; WO 95/17210). Suitably, the metabolisable
oil is present in an amount
of 0.5% to 20% (final concentration) of the total volume of the immunogenic
composition, suitably an
amount of 1.0% to 10% of the total volume, suitably in an amount of 2.0% to
6.0% of the total volume.
In a specific embodiment, the metabolisable oil is present in a final amount
of about 0.5%, 1%,
3.5% or 5% of the total volume of the immunogenic composition. In another
specific embodiment, the
metabolisable oil is present in a final amount of 0.5%, 1%, 3.57% or 5% of the
total volume of the
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immunogenic composition. A suitable amount of squalene is about 10.7 mg per
dose, suitably from 10.4
to 11.0 mg per dose.
Suitably the oil-in-water emulsion systems used in the present invention have
a small oil droplet
size in the sub-micron range. Suitably the droplet sizes will be in the range
120 to 750 nm, suitably sizes
from 120 to 600 nm in diameter. Typically the oil-in water emulsion contains
oil droplets of which at least
70% by intensity are less than 500 nm in diameter, in particular at least 80%
by intensity are less than
300 nm in diameter, suitably at least 90% by intensity are in the range of 120
to 200 nm in diameter.
The oil-in-water emulsion according to the invention may comprise a sterol or
a tocopherol, such
as alpha-tocopherol. Sterols are well known in the art, for example
cholesterol is well known and is, for
example, disclosed in the Merck Index, 11th Edition, page 341, as a naturally
occurring sterol found in
animal fat. Other suitable sterols include P-sitosterol, stigmasterol,
ergosterol and ergocalciferol. Suitably
alpha-tocopherol or a derivative thereof, such as alpha-tocopherol succinate
is present. Suitably alpha-
tocopherol is present in an amount of between 0.2% and 5.0% (v/v) of the total
volume of the
immunogenic composition, suitably at an amount of 2.5% (v/v) in a 0.5 ml dose
volume, or 0.5% (v/v) in
0.5 ml dose volume or 1.7-1.9% (v/v), suitably 1.8% in 0.7 ml dose volume. By
way of clarification,
concentrations given in v/v can be converted into concentration in w/v by
applying the following conversion
factor: a 5% (v/v) alpha-tocopherol concentration is equivalent to a 4.8%
(w/v) alpha-tocopherol
concentration. A suitable amount of alpha-tocopherol is about 11.9 mg per
dose, suitably from 11.6 to 12.2
mg per dose.
The oil-in-water emulsion may comprise an emulsifying agent. The emulsifying
agent may be
present at an amount of 0.01 to 5.0% by weight of the immunogenic composition
(w/w), suitably present
at an amount of 0.1 to 2.0% by weight (w/w). Suitable concentration are 0.5 to
1.5% by weight (w/w) of
the total composition. The emulsifying agent may suitably be polyoxyethylene
sorbitan monooleate
(polysorbate 80 or Tween 80). In a specific embodiment, a 0.5 ml dose volume
contains 1% (w/w) Tween
80, and a 0.7 ml dose volume contains 0.7% (w/w) Tween 80. In another specific
embodiment the
concentration of Tween 80 is 0.2% (w/w). A suitable amount of polysorbate 80
is about 4.9 mg per dose,
suitably from 4.6 to 5.2 mg per dose.
Span 85 (polyoxyethylene sorbitan trioleate) may also be present, for example
at a level of 1%.
An example of oil-in-water emulsion adjuvant comprises Span85 for use in the
invention is given and
detailed in EP0399843B, also known as MF59.
In a preferred embodiment, the adjuvant is an oil-in-water emulsion comprising
squalene, alpha-
tocopherol and a surfactant, i.e. the emulsion comprises squalene, alpha-
tocopherol and a surfactant, e.g.
polysorbate 80, in an aqueous phase. Preparation of such adjuvants is e.g.
described in WO 95/17210 and
WO 06/100109. In one embodiment, the emulsion comprises 2-10% (v/v) squalene,
from 2-10% (v/v)
alpha-tocopherol and from 0.3-3% (v/v) Tween 80. Preferably, the emulsion
comprises 2.5% squalene
(v/v), 2.5% alpha-tocopherol (v/v), 0.9% polyoxyethylene sorbitan monooleate
(v/v) (Tween 80).
Autoimmune diseases to be prevented and/or treated
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As explained above, the invention relates to the use of adjuvants for the
prevention and/or
treatment of autoimmune diseases. The following diseases have been classified
as autoimmune diseases:
Acute disseminated encephalomyelitis, Addison's disease, Agammaglobulinemia,
Alopecia areata,
Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Anti
phospholipid syndrome (APS),
Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Axonal & neuronal
neuropathy (AMAN),
Behcet's disease, Bullous pemphigoid, Castleman disease (CD), Celiac disease,
Chagas disease, Chronic
inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal
osteomyelitis (CRMO),
Churg-Strauss, Cicatricial pemphigoid/benign mucosa! pemphigoid, Cogan's
syndrome, Cold agglutinin
disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome,
Crohn's disease, Dermatitis
herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica),
Discoid lupus, Dressler's syndrome,
Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis,
Erythema nodosum, Essential mixed
cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant
cell arteritis (temporal arteritis),
Giant cell myocarditis, Glomerulonephritis, Good pasture's syndrome,
Granulomatosis with Polyangiitis,
Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolytic
anemia, Henoch-Schonlein
purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG),
Hypogammalglobulinemia, IgA
Nephropathy, IgG4-related sclerosing disease, Inclusion body myositis (IBM),
Interstitial cystitis (IC),
Juvenile arthritis, Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton
syndrome, Leukocytoclastic
vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear
IgA disease (LAD), Lupus, Lyme
disease chronic, Meniere's disease, Microscopic polyangiitis (MPA), Mixed
connective tissue disease (MCTD),
Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis (MS), Myasthenia
gravis, Myositis,
Narcolepsy, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid,
Optic neuritis, Palindromic
rheumatism (PR), PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders
Associated with
Streptococcus), Paraneoplastic cerebellar degeneration (PCD), Paroxysmal
nocturnal hemoglobinuria
(PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonnage-
Turner syndrome,
Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious
anemia (PA), POEMS
syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,
skin changes),
Polyarteritis nodosa, Polymyalgia rheumatic, Polymyositis, Postmyocardial
infarction syndrome,
Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing
cholangitis, Progesterone
dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA),
Pyoderma gangrenosum, Raynaud's
phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's
syndrome, Relapsing
polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis,
Rheumatic fever, Rheumatoid
arthritis (RA), Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma,
Sjogren's syndrome, Sperm &
testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial
endocarditis (SBE), Susac's
syndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporal
arteritis/Giant cell arteritis,
Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse
myelitis, Type 1 diabetes,
Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD),
Uveitis, Vasculitis, Vitiligo, and
Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).
Preferred disease to be prevented and/or treated according to the invention
include:
= Rheumatoid Arthritis
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In people with rheumatoid arthritis, the immune system predominantly targets
the lining (synovium)
that covers various joints. Inflammation of the synovium is usually
symmetrical (occurring equally on
both sides of the body) and causes pain, swelling, and stiffness of the
joints. These features distinguish
rheumatoid arthritis from osteoarthritis, which is a more common and
degenerative "wear-and-tear"
arthritis. Currently available therapy focuses on reducing inflammation of the
joints with anti-
inflammatory or immunosuppresssive medications. Sometimes, the immune system
may also target
the lung, blood vessels, or eye; occasionally patients may also develop
symptoms of other autoimmune
diseases such as Sjogren's the inflammation, itching, and scaling.
= Multiple Sclerosis
Multiple sclerosis is a disease in which the immune system targets nerve
tissues of the central nervous
system. Most commonly, damage to the central nervous system occurs
intermittently, allowing a person
to lead a fairly normal life. At the other extreme, the symptoms may become
constant, resulting in a
progressive disease with possible blindness, paralysis, and premature death.
Some medications such
as beta interferon are helpful to people with the intermittent form of
multiple sclerosis. In young adults,
multiple sclerosis is the most common disabling disease of the nervous system.
= Immune-Mediated or Type 1 Diabetes Mellitus
Type 1 diabetes mellitus results from autoimmune destruction of the insulin-
producing cells of the
pancreas. Insulin is required by the body to keep the blood sugar (glucose)
level under control. High
levels of glucose are responsible for the symptoms and the complications of
the disease. However,
most of the insulin-producing cells are destroyed before the patient develops
symptoms of diabetes.
Symptoms include fatigue, frequent urination, increased thirst, and possible
sudden confusion. Type 1
diabetes mellitus is usually diagnosed before the age of 30 and may be
diagnosed as early as the first
month of life. Together with Type 2 diabetes (not considered an autoimmune
disease), diabetes
mellitus is the leading cause of kidney damage, loss of eyesight, and leg
amputation. Close control of
sugar levels decreases the rate at which these events occur.
= Inflammatory Bowel Diseases
This medical term is used for both Crohn's disease and ulcerative colitis, two
diseases in which the
immune system attacks the gut (intestine). Patients may have diarrhea, nausea,
vomiting, abdominal
cramps, and pain that can be difficult to control. Illness in afflicted
individuals can result from intestinal
inflammation and from side effects of the drugs used for the disease. For
example, daily use of high-
dose corticosteroid (prednisone) therapy, which is needed to control severe
symptoms of Crohn's
disease, can predispose patients to infections, bone thinning (osteoporosis),
and fractures.
= Systemic Lupus Erythematosus
Patients with systemic lupus erythematosus most commonly experience profound
fatigue, rashes, and
joint pains. In severe cases, the immune system may attack and damage several
organs such as the
kidney, brain, or lung. For many individuals, symptoms and damage from the
disease can be controlled
with available anti-inflammatory medications.
= Psoriasis
Psoriasis is an immune system disorder that affects the skin, and occasionally
the eyes, nails, and
joints. Psoriasis may affect very small areas of skin or cover the entire body
with a buildup of red scales
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called plaques. The plaques are of different sizes, shapes, and severity and
may be painful as well as
unattractive. Bacterial infections and pressure or trauma to the skin can
aggravate psoriasis. Most
treatments focus on topical skin care to relieve the inflammation, itching,
and scaling.
= Scleroderma
This autoimmune disease results in thickening of the skin and blood vessels.
Almost every patient with
scleroderma has Raynaud's, which is a spasm of the blood vessels of the
fingers and toes. Symptoms
of Raynaud's include increased sensitivity of the fingers and toes to the
cold, changes in skin color,
pain, and occasionally ulcers of the fingertips or toes. In people with
scleroderma, thickening of skin
and blood vessels can result in loss of movement and shortness of breath or,
more rarely, in kidney,
heart, or lung failure.
= Autoimmune Thyroid Diseases
Hashimoto's thyroiditis and Grave's disease result from immune system
destruction or stimulation of
thyroid tissue. Symptoms of low (hypo-) or overactive (hyper-) thyroid
function are nonspecific and
can develop slowly or suddenly; these include fatigue, nervousness, cold or
heat intolerance, weakness,
changes in hair texture or amount, and weight gain or loss. The diagnosis of
thyroid disease is readily
made with appropriate laboratory tests. The symptoms of hypothyroidism are
controlled with
replacement thyroid hormone pills; however, complications from over- or under-
replacement of the
hormone can occur. Treatment of hyperthyroidism requires long-term anti-
thyroid drug therapy or
destruction of the thyroid gland with radioactive iodine or surgery. Both of
these treatment approaches
carry certain risks and long-term side effects.
In a preferred embodiment, the autoimmune disease to be prevented and/or
treated is a disease
affecting the nervous system, such as the central nervous system. In an even
more preferred embodiment,
the autoimmune disease is multiple sclerosis. In a further more preferred
embodiment, the autoimmune
disease it type 1 diabetes. In a further preferred embodiment, the autoimmune
disease is acute
disseminated encephalomyelitis.
In particular, adjuvants comprising an oil-in-water emulsion, e.g. comprising
squalene, alpha-
tocopherol and polysorbate 80 in an aqueous phase, are preferred for the
prevention and/or treatment of
diseases affecting the nervous system, such as the central nervous system,
e.g. multiple sclerosis.
Treatment options
In preferred embodiments, the method of the invention, or use of the
invention, comprises multiple
administrations of the adjuvant, for example, at least 2, at least 3, at least
4, at least 5, or at least 10
administrations of the adjuvant. In one embodiment, the time interval between
each of the administrations
is between 1 day and 6 months, e.g. the interval may be from 1 week to 1 month
between each
administration.
In a preferred embodiment, the subject is a human subject. The human subject
to be treated using
the method of the invention may be of any age. In one embodiment, however, the
human subject is more
than 18 years of age when the treatment is initiated. In further embodiments,
the subject is more than 40
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years of age, such as more than 50 years of age, e.g. more than 60 years of
age. The subject may be male
or female.
In one embodiment, the adjuvant is for use in the prevention of an autoimmune
disease in a human
subject who is pre-disposed to develop an autoimmune disease, for example, a
subject who is pre-disposed
to develop multiple sclerosis or a subject who is pre-disposed to develop type
1 diabetes. A human subject
who is pre-disposed to develop an autoimmune disease can, for example, be a
subject who shows early
clinical signs of an autoimmune disease, a subject who has a genetic risk of
developing an autoimmune
disease or a subject who has specific auto-antibodies.
A person pre-disposed to develop multiple sclerosis is e.g. a person having
CIS (clinically isolated
syndrome) ¨ a first and single neurologic episode of inflammation or
demyelination in the central nervous
system lasting at least 24 hours, in particular when lesions consistent with
multiple sclerosis are seen on
MRI.
A person pre-disposed to develop type 1 diabetes is e.g. a person having auto-
antibodies, such as
autoantibodies GADA, IA-2A and/or mIAA (Sosenko etal. (2013) Diabetes Care 36:
2615).
The adjuvant may be administered via various suitable routes, including
parenteral, such as
intramuscular, intradermal or subcutaneous administration. Suitably, the
adjuvant compositions used in the
present invention have a human dose volume of between 0.05 ml and 1 ml, such
as between 0.1 and 0.5
ml, in particular a dose volume of about 0.5 ml, or 0.7 ml.
The adjuvant may be given as monotherapy or in combination with other
substances, e.g. other
substances known to have a therapeutic or prophylactic effect on autoimmune
diseases.
The teachings of all references in the present application, including patent
applications and granted
patents, are herein fully incorporated by reference. The terms 'comprising',
'comprise' and 'comprises'
herein are optionally substitutable with the terms 'consisting of', 'consist
of', and 'consists of', respectively.
The invention will be further described by reference to the following, non-
limiting, examples:
EXAMPLES
Example 1 - Methods
Mice
WT mice were purchased from Janvier Labs (France). 2D2 T-cell receptor
transgenic mice, specific for
myelin oligodendrocyte glycoprotein, were purchased from the Jackson
Laboratory. Foxp3-IRES-GFP knock-
in (F0xp3GFP) mice were kindly provided by Pr. Bernard Malissen. 2D2 and
F0xp3GFP mice were
backcrossed with CD90.1 congenic animals. All mice were on a C57131/6
background. Mice were housed
under specific pathogen-free conditions and were studied at 7-14 week of age.
All experimental protocols
were approved by the local ethics committee and are in compliance with
European Union guidelines.
Reagents
AS01 is composed of MPL (1 mg/ml), QS-21 (1 mg/ml) and liposomes. A503 is
composed of alpha-
tocopherol (23,72 mg/ml), squalene oil (21.38 mg/ml) and polysorbate 80 (9,72
mg/ml). A504 is composed
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of MPL (0.1 mg/ml) and alum (1 mg/ml). Freund's adjuvants were purchased from
Sigma-Aldrich.
Incomplete form (IFA) contains 85% of paraffin oil and 15% of mannide
monooleate. Complete form (CFA)
adds Mycobacterium tuberculosis (H37Ra) heat killed and dried at a
concentration of 1 mg/ml.
Administration of adjuvants
Mice received 30 pl of vaccine adjuvant or PBS by subcutaneous route in left
hind footpads. To analyze
their effect, left popliteal draining lymph nodes (dLN) and non-draining right
brachial lymph nodes were
collected at different time points. In mice that were immunized to induce EAE
(experimental auto-immune
encephalomyelitis), they received 100 pl of vaccine adjuvant or PBS by
subcutaneous route at the base of
the tail and at the upper back 3 days before and on the day of EAE
(experimental autoimmune
encephalomyelitis) induction.
Flow cytometry analyses
Cells from lymph nodes were mechanically dissociated and resuspended in PBS 3%
SVF. They were first
treated with 2.4G2 antibody to block the Fc receptor and stained with the
following antibodies: anti-CD45
(30-F11), anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25
(PC61), anti-Foxp3
(MF23), anti-CTLA-4 (UC10-4F10-11), anti-GITR (DTA-1), anti-ICOS (17G9), anti-
Ki-67 (B56). All antibodies
were obtained from BD Biosciences. Intracellular staining was performed using
the Intracellular Fixation &
Permeabilization Buffer Set from eBioscience. Cells were acquired on a BD
LSRII cytometer and analyzed
using Flowio software.
Treg peripheral induction assessment
Lymph nodes (brachial, axillary, cervical, and inguinal) and spleen cells of
Foxp3GFP CD90.1 mice were
isolated, treated as above and stained with an anti-CD4 antibody. CD4+GFP-
cells (Tconv) were purified
using a BD FACSAria II then intravenously injected in WT mice (106
cells/mouse). The following day, mice
received 30 pl of vaccine adjuvant or PBS by subcutaneous route in left hind
footpads. Three days after,
dLN were collected and cells were stained with anti-CD4, anti-CD90.1 (OX-7),
anti-CD90.2 (30-H12) and
anti-Foxp3 antibodies. Foxp3 induction in CD90.1 transferred cells was
evaluated by flow cytometry.
In vitro Treg suppression assay
Foxp3GFP mice received 100 pl of vaccine adjuvant or PBS by subcutaneous route
at the base of the tail
and at the upper back. Brachial and inguinal lymph nodes were collected at 3
or 7 days. Cells were isolated
and stained as described above. CD4+GFP- cells (Tconv) and CD4+GFP+ cells
(Treg) were then sorted using
BD FACSAria II. Tconv cells were labeled with CellTrace Violet Proliferation
Kit (Life technologies) and
plated in 96-well plate at 2.5 x 104 cells/well with splenocytes from CD3-/-
mice at 7.5 x 104 cells/well.
Culture medium was supplemented with anti-CD3 (KT3) from BioXCell at 0.05
pg/ml. Treg cells were then
added at different ratios from 1:1 to 1:16. At day 3, CellTrace dilution was
assessed by flow cytometry.
EAE induction
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Mice were immunized by subcutaneous injection of 100 pg of M0G35-55 peptide
(PolyPeptide) emulsified in
100 pl of CFA (Sigma-Aldrich) supplemented with 50 pg of heat-killed
Mycobacterium tuberculosis H37Ra
(BD Biosciences). Animals were additionally injected intravenously with 200 ng
of Bordetella pertussis toxin
(Enzo) at the time of, and two days following immunization. The clinical
evaluation was performed on a
daily basis by a 5-point scale ranging from 0, no clinical sign; 1, limp tail;
2, limp tail, impaired righting
reflex, and paresis of one limb; 3, hindlimb paralysis; 4, hindlimb and
forelimb paralysis; 5, moribund.
Cytokine measurement
Ten days after EAE induction, brachial and inguinal dLN were collected. Cells
were cultured with 1 pg/ml
of M0G35-55 peptide in 96-well plate at 2 x 105 cells/well. At day 3,
supernatants were harvested to measure
INF-y and IL-17 secretions by ELISA (eBioscience).
T cell priming evaluation
Lymph nodes and spleen cells from 2D2 CD90.1 mice were stained with the
following biotin-labeled
antibodies: anti-CD19 (6D5), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD8
(53-6.7) and anti-CD25
(7D4) and then were coated with anti-biotin microbeads (Miltenyi Biotec).
After magnetic sorting, cells of
the CD4+ enriched negative fraction were labeled with CellTrace Violet
Proliferation Kit and were
intravenously injected in naive mice (106 cells/mouse). The following day,
mice were immunized with
M0G35-55peptide as for EAE induction. CellTrace dilution of CD4+CD90.1+V[311+
(T-cell receptor transgene)
cells was assessed by flow cytometry from brachial and inguinal lymph nodes at
day 3. In mice receiving
adjuvants, they were injected with 100 pl of vaccine adjuvant or PBS by
subcutaneous route at the base
of the tail and at the upper back.
Treg cell adoptive transfer
Foxp3GFP CD90.1 mice received 100 pl of vaccine adjuvant or PBS by
subcutaneous route at the base of
the tail and at the upper back. At day 3, brachial and inguinal lymph nodes
were collected and cells were
stained with anti-CD4 antibody. CD4+GFP+ cells (Treg) were then purified using
BD FACSAria II and
intravenously injected in WT mice (1 x 106 cells/mouse). The following day,
mice were immunized with
M0G35-55 peptide as previously described in EAE induction.
Statistical analysis
Statistical analyses were performed using GraphPad Prism Software. Statistical
significance was determined
using the two-tailed nonparametric Mann¨Whitney U test. *p<0.05, **p<0.01,
***p<0.001. Means SEM
were used throughout the figures.
Example 2 - Vaccine adjuvants induce transient preferential expansion of Treg
cells
CFA, incomplete Freund's adjuvants (IFA), AS01, A503 and A504 were
administered
subcutaneously to mice. After 4 days, a massive inflammation and swelling of
the draining lymph nodes
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(dLN) was observed (Fig. la). The cellularity of these dLN was increased by 11
to 20 fold (Fig. 2a). By
contrast, injection of alum had no effect on size and cell numbers of dLN.
Treg cells were then analyzed by flow cytometry (Fig. lb). Except for alum, we
observed a rapid
increase (day 4) of Treg cell proportions among CD4+ cells after injection of
the different adjuvants, up to
1.9 fold compared to the PBS-injected mice (Fig. 1c). This Treg cell expansion
was associated with an
activated phenotype, as shown by an increased expression of CD44, ICOS and Ki-
67 (Fig. 2b). These Treg
cell expansion and activation were transient since it was no longer present at
day 7, except for IFA (Fig.
1c). Treg cell expansion driven by adjuvant injections was due to thymic Tregs
(tTregs) accumulation and
not to increased induction of peripheral Treg. Indeed, Foxp3 expression was
not induced by adjuvants in
adoptively transferred CD4+Foxp3- cells (Fig. 1d). These data revealed the in
vivo activation of tTreg cells
by new generation adjuvants.
We then assessed the effect of vaccine adjuvant administration on Treg cell
activity by classical in
vitro suppression assay. Alum, Freund's adjuvant, A503 and A504 had no effect
on Treg cell function (Fig.
2c), while AS01 partially reduced their suppressive activity and Foxp3
expression (Fig. le and if and 2d).
This effect was observed at day 4 but not anymore at day 7 (Fig. 2e). Thus,
administration of new
generation adjuvants did not impair, or only marginally, the suppressive
function of Treg cells.
Example 3 - EAE prevention by administration of AS01 and AS03 adjuvants
In order to evaluate the effects of vaccine adjuvants on tolerance induction
in a physiopathological
context, we tested adjuvant treatments in the EAE autoimmune model.
Strikingly, 2 injections of AS01 or
A503, 3 days before and at the time of immunization, induced almost complete
prevention from disease
development. Mean clinical scores were below 0.14-0.17, compared to 1.17 in
controls. In contrast, A504
and alum had no effect on EAE (Fig. 3a). After only one injection of AS01 or
AS03 performed 3 days before,
or at the time of immunization, EAE was significantly delayed or reduced,
respectively (Fig. 4a and 4b).
Thus, AS01 and A503 have strong preventing effect on EAE.
We next investigated priming and cytokine polarization of auto-reactive Tconv
cells. T-cell
proliferation was assessed after adoptive transfer of 2D2 transgenic mice
expressing a transgenic I-Ab¨
restricted T cell receptor specific for M0G35-55 peptide (Bettelli etal.
(2003) J. Exp. Med. 197: 1073) prior
to EAE induction. AS01 administration slightly reduced the proliferation of
M0G35-55-specific T cells at day
3 in dLN, whereas A503 had no effect (Fig. 3b). T-cell polarization was
assessed by measuring the release
of IFNy and IL-17, 2 major pathogenic cytokines in EAE, at day 10 in dLN after
EAE induction by auto-
reactive Tconv cells specific to the M0G35-55 immunizing peptide. AS01
administration was associated with
a decrease of IFNy production, whereas IFNy and IL-17 were unaffected by A503
administration (Fig. 3c).
Thus, although both AS01 and A503 administration prevented EAE development,
only AS01 disrupted
priming and polarization of M0G35-55 reactive T-cells.
We then performed adoptive transfer experiments to further analyze the role of
Treg cells in
adjuvant-driven EAE prevention. Purified Treg cells from dLN of adjuvant
treated mice were injected into
naive mice prior to EAE induction. Amazingly, mice that received Treg cells
from A503 treated mice were
fully protected from the disease. In contrast, Treg cells from AS01 treated
mice had no effect since
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recipients mice transferred with these cells developed the same EAE as control
mice transferred with PBS
or Treg from naive mice (Fig. 3d). These data reveal that A503 administration
strongly enhanced the
capacity of Treg cells to control EAE.
To gain more insight into the mechanism of EAE prevention by A503, we further
studied Treg cells
and molecules involved in T-cell migration. Cells from dLN of mice immunized
to induce EAE and treated
with A503 were analyzed at day 10. Treg cell proportion was significantly
increased in A503 treated mice
compare to control (Fig. 3e). The expression of the CCR6 and CXCR3 chemokine
receptors by Tconv cells,
which play a critical role in the entry of pathogenic T cells into the central
nervous system (CNS) during
EAE (Reboldi etal. (2009) Nat. Immunol. 10: 514; Sporici and Issekutz (2010)
Eur. J. Immunol. 40: 2751),
was unaffected by the A503 treatment (Fig. 4c). Interestingly, the expression
levels of integrins involved
in CNS homing (Yednock etal. (1992) Nature 356: 63; Rothhammer etal. (2011)].
Exp. Med. 208: 2465)
were significantly modified by the adjuvant. Treg cells expressed a higher
level of integrin aL while Tconv
cells showed a decreased level of integrin aM (Fig. 3f). Moreover, expression
levels of integrins a4 and aL
by Tconv cells were unaffected (Fig. 4d). These results suggest that A503
administration impacts on
migration of Treg and Tconv cells in the CNS, which may explain its capacity
to suppress EAE.
Conclusion
This work demonstrates that some of the new generation vaccine adjuvants have
strong immuno-
regulatory properties and impact on Treg cell activation and function, thus
revealing their potential in the
treatment of autoimmune disorders. Both AS01 and A503 administration induced
protection from EAE.
Interestingly, their suppressive mechanism appears different. AS01 altered
priming and cytokine
polarization of encephalitogenic Tconv cells whereas A503 strongly increased
the protective capacity of
Treg cells in EAE and may modify the migration of Tconv and Treg cells.
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